Selection-free zinc-finger-nuclease engineering by context-dependent assembly (CoDA)

Abstract

Engineered zinc-finger nucleases (ZFNs) enable targeted genome modification. Here we describe context-dependent assembly (CoDA), a platform for engineering ZFNs using only standard cloning techniques or custom DNA synthesis. Using CoDA-generated ZFNs, we rapidly altered 20 genes in Danio rerio, Arabidopsis thaliana and Glycine max. The simplicity and efficacy of CoDA will enable broad adoption of ZFN technology and make possible large-scale projects focused on multigene pathways or genome-wide alterations.

Figure 1. Schematic overview of Context-Dependent Assembly (CoDA)

Zinc fingers are represented as colored spheres (F1 = amino-terminal finger, F2 = middle finger, F3 = carboxy-terminal finger) and 3 bp DNA “subsites” are represented as colored rectangles. Two different three-finger arrays, each engineered to bind different 9 bp target sites and that each share a common middle F2 can be used to create a three-finger array with a new specificity by joining together the amino-terminal finger (F1) from the first array, the middle finger common to both arrays (F2), and the carboxy-terminal finger (F3) from the second array.